Characterization of African horsesickness virus serotype 4-induced polypeptides in Vero cells and their reactivity in Western immunoblotting

Abstract

The structural and non-structural proteins induced by African horsesickness virus serotype 4 (AHSV-4) in infected Vero cells were analysed by SDS-PAGE. Twenty-two virus-induced polypeptides were detected in infected cells by comparison with the polypeptides of mock-infected cells, of which four major (VP2, VP3, VP5 and VP7) and three minor (VP1, VP4 and VP6) structural proteins and four non-structural proteins (P58, P48, P21 and P20) were shown to be virus-coded, as deduced from electrophoretic and antigenic studies of purified virions and infected cells. The proteins that elicit the major antibody responses both in vaccinated and naturally or experimentally infected horses were shown to be three structural proteins, VP2, VP5 and VP7, and the four major non-structural proteins, P58, P48, P21 and P20, as deduced by radioimmunoprecipitation and immunoblotting assays. The crossreactivity between AHSV-4 and sera obtained from horses experimentally infected with seven other serotypes was also determined. The results showed that VP5, VP7, P48, P21 and P20 are conserved and can be used to diagnose the infection of any of these eight serotypes.

Purification and properties of virus particles, infectious subviral particles, cores and VP7 crystals of African horsesickness virus serotype 9

“…We have observed in purified virus three distinct forms of VP5 with different migration rates in SDS-PAGE (VP5, VP5' and VP5"), which after recovery and re-analysis by SDS-PAGE all comigrate and therefore appear to be of similar M r. It is considered likely that VP5 can adopt different conformational structures which are at least partially dependent on the reducing environment used and which alter its rate of migration during SDS PAGE. Laviada et al (1993) also detected a second protein band in the region of VP5 when purified particles of AHSV-4 were analysed by SDS-PAGE. They confirmed its relatedness to VP5 by its reaction with a VP5-specific monoclonal antibody.…”

Expression of nonstructural protein NS3 of African horsesickness virus (AHSV): evidence for a cytotoxic effect of NS3 in insect cells, and characterization of the gene products in AHSV infected Vero cells

“…Two related protein products were synthesized from AHSV S10 in in vitro translation studies, and their counterparts identified in AHSV infected cells. These proteins were termed NS3 and NS3A in accordance with BTV nomenclature [37], but they have also been described as NS4 and NS4a [12], and P21 and P20 [20]. Based on AHSV genome coding assignments (R. O'Hara, pers.…”

“…proteins are synthesized in infected cells [4,12,20,26]. The gene products of the smallest genome segment of BTV, segment 10 (S10), are the minor nonstructural proteins NS3 and NS3A.…”

Development of a Luminex-Based DIVA Assay for Serological Detection of African Horse Sickness Virus in Horses

“…The analysis of serum samples from uninfected, experimentally infected and vaccinated horses suggests that the assay offers high sensitivity, specificity and reproducibility (Table ). VP7 was selected as a viral antigen because it is the main target protein for group‐specific diagnosis (OIE, ), while NS3, the second most variable AHSV protein, was selected because it is not expressed in horses vaccinated with inactivated AHSV4 vaccine, allowing DIVA diagnosis (Laviada et al., ). All animals vaccinated with inactivated AHSV4 vaccine in our study were positive for VP7 and negative for NS3 (Fig.…”

“…To avoid these drawbacks, new vaccines, such as killed vaccines, DNA vaccines or recombinant vaccines, have been developed as an alternative to attenuated vaccines (Laviada et al., ; Guthrie et al., ; Alberca et al., ), and this new generation of vaccines enables the development of diagnostic methods that differentiate infected from vaccinated animals (DIVA). One example of these DIVA vaccines is a purified, inactivated AHSV4 vaccine that does not induce antibodies against NS3 (Laviada et al., , ). The use of such vaccines, combined with a reliable DIVA test against AHS that can simultaneously detect antibodies against different AHSV antigens in a single sample, would allow the rapid assessment of whether individual horses have not been infected, have been infected or have been vaccinated.…”